Characterization of novel endo-ß-N-acetylglucosaminidases from intestinal Barnesiella intestinihominis that hydrolyze multi-branched complex-type N-glycans.

Endo-ß-N-acetylglucosaminidases (ENGases) are enzymes that hydrolyze N-linked glycans. Many ENGases have been characterized, but few have been identified with hydrolytic activity towards multi-branched complex-type N-glycans. In this study, three candidate ENGases were identified from Barnesiella intestinihominis based on database searches and phylogenetic analysis. A domain search identified the N x E motif in all three candidates, suggesting that they were members of glycosyl hydrolase family 85 (GH85). The three candidate ENGases, named Endo-BIN1, Endo-BIN2, and Endo-BIN3, were expressed in Escherichia coli cells, and their hydrolytic activity towards N-glycans and glycoproteins was measured by high performance liquid chromatography analysis and SDS-PAGE analysis. All ENGases showed hydrolytic activity towards glycoproteins, but only Endo-BIN2 and Endo-BIN3 showed hydrolytic activity towards pyridylaminated N-glycans. The optimum pH of Endo-BIN1, Endo-BIN2, and End-BIN3 was pH 6.5, 4.0, and 7.0, respectively. We measured substrate specificities of Endo-BIN2 and Endo-BIN3 towards pyridylaminated N-glycans, and found that the two Endo-BIN enzymes showed similar substrate specificity, preferring bi-antennary complex-type N-glycans with galactose or α2,6-linked sialic acid residues at the non-reducing ends. Endo-BIN2 and Endo-BIN3 were also able to hydrolyze multi-branched complex-type N-glycans. SDS-PAGE analysis revealed that all Endo-BIN enzymes were capable of releasing complex-type N-glycans from glycoproteins such as rituximab, transferrin, and fetuin. We expect that B. intestinihominis possesses ENGases to facilitate the utilization of complex-type N-glycans from host cells. These findings will have applications in N-glycan remodeling of glycoproteins and the development of pharmaceuticals.

ACE2 N-glycosylation modulates interactions with SARS-CoV-2 spike protein in a site-specific manner.

SARS-CoV-2 has evolved continuously and accumulated spike mutations with each variant having a different binding for the cellular ACE2 receptor. It is not known whether the interactions between such mutated spikes and ACE2 glycans are conserved among different variant lineages. Here, we focused on three ACE2 glycosylation sites (53, 90 and 322) that are geometrically close to spike binding sites and investigated the effect of their glycosylation pattern on spike affinity. These glycosylation deletions caused distinct site-specific changes in interactions with the spike and acted cooperatively. Of note, the particular interaction profiles were conserved between the SARS-CoV-2 parental virus and the variants of concern (VOCs) Delta and Omicron. Our study provides insights for a better understanding of the importance of ACE2 glycosylation on ACE2/SARS-CoV-2 spike interaction and guidance for further optimization of soluble ACE2 for therapeutic use.

Recombinant lectin Gg for brain imaging of glycan structure and formation in the CNS node of Ranvier.

The nodes of Ranvier are unmyelinated gaps in the axon, important for the efficient transmission of action potentials. Despite the identification of several glycoproteins involved in node formation and maintenance, glycans' structure and formation in the node remain unclear. Previously, we developed a recombinant lectin from the Clostridium botulinum neurotoxin complex, specific to the galactose and N-acetylgalactosamine terminal epitopes (Gg). Gg stained Neuro2a cells. Here, we show Gg punctuate staining in mouse brain cryosections. Thus, we hypothesized that Gg could help study glycans in the node of Ranvier. Lectin histochemistry on mouse brain cryosections confirmed that Gg binds specifically to the node of Ranvier in the central nervous system (CNS). Using a combination of lectin blotting, glycosidase treatment on tissue sections, and lectin histochemistry, Gg ligands were identified as α-galactose terminal glycoproteins in the perinodal extracellular matrix. Furthermore, we detected the spatiotemporal distribution of galactosylated glycans in the CNS node of Ranvier in mouse brain tissues at different postnatal times. Finally, we observed impaired clustering of galactosylated glycans in the nodes during demyelination and remyelination in cuprizone-induced demyelination and remyelination mouse model. In conclusion, Gg can serve as a novel brain imaging tool in glycobiology and report glycoprotein formation and alterations in the CNS node of Ranvier. Our findings might serve as a first step to establish the role of glycans in the node of Ranvier.

Preparation of Glycan Arrays Using Glycopeptides Derived From Biomaterials.

In general, viruses recognize host cell surface glycans, but the measurement of virus-host cell glycan interaction is not widely operated. This is not only because commercially available, structure-defined glycans are limited, but also because such interactions, if any, between viruses and isolated glycans are relatively weak, and thus, difficult to detect by conventional methods, e.g., enzyme-linked immune-sorbent assay. We describe a practical method to detect virus binding to glycans; for this, preparation of glycan arrays using glycopeptides derived from biomaterials is necessary. In this context, neoglycoprotein is produced using bovine serum albumin (BSA) and commercially available glycopeptides, with which influenza viruses are detected using an evanescent-field-activated fluorescence scanner. It is clearly shown that H1N1 strains of influenza virus recognize BSA, to which DiNeuα2-6bianntena-peptide (SGP) is covalently linked, while on the other hand H5N1 strains recognize BSA linked to DiNeuα2-3bianntena-peptide (α2,3SGP).

Significant role of host sialylated glycans in the infection and spread of severe acute respiratory syndrome coronavirus 2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been transmitted across all over the world, in contrast to the limited epidemic of genetically- and virologically-related SARS-CoV. However, the molecular basis explaining the difference in the virological characteristics among SARS-CoV-2 and SARS-CoV has been poorly defined. Here we identified that host sialoglycans play a significant role in the efficient spread of SARS-CoV-2 infection, while this was not the case with SARS-CoV. SARS-CoV-2 infection was significantly inhibited by α2-6-linked sialic acid-containing compounds, but not by α2-3 analog, in VeroE6/TMPRSS2 cells. The α2-6-linked compound bound to SARS-CoV-2 spike S1 subunit to competitively inhibit SARS-CoV-2 attachment to cells. Enzymatic removal of cell surface sialic acids impaired the interaction between SARS-CoV-2 spike and angiotensin-converting enzyme 2 (ACE2), and suppressed the efficient spread of SARS-CoV-2 infection over time, in contrast to its least effect on SARS-CoV spread. Our study provides a novel molecular basis of SARS-CoV-2 infection which illustrates the distinctive characteristics from SARS-CoV.

Characterization of novel endo-ß-N-acetylglucosaminidase from Bacteroides nordii that hydrolyzes multi-branched complex type N-glycans.

Endo-ß-N-acetylglucosaminidases (ENGases) are enzymes that hydrolyze the N-linked oligosaccharides. Many ENGases have already been identified and characterized. However, there are still a few enzymes that have hydrolytic activity toward multibranched complex-type N-glycans on glycoproteins. In this study, one novel ENGase from Bacteroides nordii (Endo-BN) species was identified and characterized. The recombinant protein was prepared and expressed in Escherichia coli cells. This Endo-BN exhibited optimum hydrolytic activity at pH 4.0. High performance liquid chromatography (HPLC) analysis showed that Endo-BN preferred core-fucosylated complex-type N-glycans, with galactose or α2,6-linked sialic acid residues at their non-reducing ends. The hydrolytic activities of Endo-BN were also tested on different glycoproteins from high-mannose type to complex-type oligosaccharides. The reaction with human transferrin, fetuin, and α1-acid glycoprotein subsequently showed that Endo-BN is capable of releasing multi-branched complex-type N-glycans from these glycoproteins.

Glycan detecting tools developed from the Clostridium botulinum whole hemagglutinin complex.

Lectins are proteins with the ability to recognize and bind to specific glycan structures. These molecules play important roles in many biological systems and are actively being studied because of their ability to detect glycan biomarkers for many diseases. Hemagglutinin (HA) proteins from Clostridium botulinum type C neurotoxin complex; HA1, HA2, and HA3 are lectins that aid in the internalization of the toxin complex by binding to glycoproteins on the cell surface. HA1 mutants have been previously reported, namely HA1 W176A/D271F and HA1 N278A/Q279A which are specific to galactose (Gal)/N-acetylgalactosamine (GalNAc) and N-acetylneuraminic acid (Neu5Ac) sugars, respectively. In this study, we utilized HA1 mutants and expressed them in complex with HA2 WT and HA3 WT to produce glycan detecting tools with high binding affinity. Particularly, two types were made: Gg and Rn. Gg is an Alexa 488 conjugated lectin complex specific to Gal and GalNAc, while Rn is an Alexa 594 conjugated lectin complex specific to Neu5Ac. The specificities of these lectins were identified using a glycan microarray followed by competitive sugar inhibition experiments on cells. In addition, we confirmed that Gg and Rn staining is clearly different depending on cell type, and the staining pattern of these lectins reflects the glycans present on the cell surface as shown in enzyme treatment experiments. The availability of Gg and Rn provide us with new promising tools to study Gal, GalNAc, and Neu5Ac terminal epitopes which can aid in understanding the functional role of glycans in physiological and pathological events.

Preparation and Detection of Glycan-Binding Activity of Influenza Virus.

We describe a method to detect influenza virus using an evanescent-field-activated fluorescence scanner type glycan array and ELISA system. Neoglycoprotein was prepared by combination of organic chemistry and biomaterial preparation. These ligands were spotted on a glass plate or plastic well to make a glycan array and ELISA plate. We detected cultured influenza virus using glycan array and ELISA. Then, we showed that the neoglycoprotein binds to Cy3-labeled hemagglutinins (H1 and H5), a NeuAcα2,6LacNAc or NeuAcα2,3LacNAc recognized protein, as detected.

Structures of human galectin-10/monosaccharide complexes demonstrate potential of monosaccharides as effectors in forming Charcot-Leyden crystals.

The galectins are a family of ß-galactoside-specific animal lectins, and have attracted much attention as novel regulators of the immune system. Galectin-10 is well-expressed in eosinophils, and spontaneously forms Charcot-Leyden crystals (CLCs), during prolonged eosinophilic inflammatory reactions, which are frequently observed in eosinophilic diseases. Although biochemical and structural characterizations of galectin-10 have been done, its biological role and molecular mechanism are still unclear, and few X-ray structures of galectin-10 in complex with monosaccharides/oligosaccharides have been reported. Here, X-ray structures of galectin-10 in complexes with seven monosaccharides are presented with biochemical analyses to detect interactions of galectin-10 with monosaccharides/oligosaccharides. Galectin-10 forms a homo-dimer in the face-to-face orientation, and the monosaccharides bind to the carbohydrate recognition site composed of amino acid residues from two galectin-10 molecules of dimers, suggesting that galectin-10 dimer likely captures the monosaccharides in solution and in vivo. d-Glucose, d-allose, d-arabinose, and D-N-acetylgalactosamine bind to the interfaces between galectin-10 dimers in crystals, and they affect the stability of molecular packing in crystals, leading to easy-dissolving of CLCs, and/or inhibiting the formation of CLCs. These monosaccharides may serve as effectors of G10 to form CLCs in vivo.

Disruption of the Dimer-Dimer Interaction of the Mumps Virus Attachment Protein Head Domain, Aided by an Anion Located at the Interface, Compromises Membrane Fusion Triggering.

Mumps virus (MuV), an enveloped negative-strand RNA virus belonging to the family Paramyxoviridae, enters the host cell through membrane fusion mediated by two viral envelope proteins, an attachment protein hemagglutinin-neuraminidase (MuV-HN) and a fusion (F) protein. However, how the binding of MuV-HN to glycan receptors triggers membrane fusion is not well understood. The crystal structure of the MuV-HN head domain forms a tetramer (dimer of dimers) like other paramyxovirus attachment proteins. In the structure, a sulfate ion (SO42-) was found at the interface between two dimers, which may be replaced by a hydrogen phosphate ion (HPO42-) under physiological conditions. The anion is captured by the side chain of a positively charged arginine residue at position 139 of one monomer each from both dimers. Substitution of alanine or lysine for arginine at this position compromised the fusion support activity of MuV-HN without affecting its cell surface expression, glycan-receptor binding, and interaction with the F protein. Furthermore, the substitution appeared to affect the tetramer formation of the head domain as revealed by blue native-PAGE analysis. These results, together with our previous similar findings with the measles virus attachment protein head domain, suggest that the dimer-dimer interaction within the tetramer may play an important role in triggering membrane fusion during paramyxovirus entry.IMPORTANCE Despite the use of effective live vaccines, mumps outbreaks still occur worldwide. Mumps virus (MuV) infection typically causes flu-like symptoms and parotid gland swelling but sometimes leads to orchitis, oophoritis, and neurological complications, such as meningitis, encephalitis, and deafness. MuV enters the host cell through membrane fusion mediated by two viral proteins, a receptor-binding attachment protein, and a fusion protein, but its detailed mechanism is not fully understood. In this study, we show that the tetramer (dimer of dimers) formation of the MuV attachment protein head domain is supported by an anion located at the interface between two dimers and that the dimer-dimer interaction plays an important role in triggering the activation of the fusion protein and causing membrane fusion. These results not only further our understanding of MuV entry but provide useful information about a possible target for antiviral drugs.

Electrical Biosensing at Physiological Ionic Strength Using Graphene Field-Effect Transistor in Femtoliter Microdroplet.

Graphene has strong potential for electrical biosensing owing to its two-dimensional nature and high carrier mobility which transduce the direct contact of a detection target with a graphene channel to a large conductivity change in a graphene field-effect transistor (G-FET). However, the measurable range from the graphene surface is highly restricted by Debye screening, whose characteristic length is less than 1 nm at physiological ionic strength. Here, we demonstrated electrical biosensing utilizing the enzymatic products of the target. We achieved quantitative measurements of a target based on the site-binding model and real-time measurement of the enzyme kinetics in femtoliter microdroplets. The combination of a G-FET and microfluidics, named a "lab-on-a-graphene-FET", detected the enzyme urease with high sensitivity in the zeptomole range in 100 mM sodium phosphate buffer. Also, the lab-on-a-graphene-FET detected the gastric cancer pathogen Helicobacter pylori captured at a distance greater than the Debye screening length from the G-FET.

N-glycan structures of human alveoli provide insight into influenza A virus infection and pathogenesis.

The rapidly evolvable influenza A virus has caused pandemics linked to millions of deaths in the past century. Influenza A viruses are categorized by H (hemagglutinin; HA) and N (neuraminidase; NA) proteins expressed on the viral envelope surface. Analyses of past pandemics suggest that the HA gene segment comes from a nonhuman virus, which is then introduced into an immunologically naïve human population with potentially devastating consequences. As a prerequisite for infection, the nonhuman HA molecules of H1-H16 viruses must be able to bind to specific sialyl receptors on the host cell surface along the human respiratory tract. Thus, additional insight into the structures of host cell glycans and how different HAs interact with different glycans might provide new insight into the mechanisms underlying sustained infection and transmission in humans. In this work, we identified the sialyl N-glycans found in normal human alveoli and characterized the influenza viruses that preferentially bound to these different structures. We also determined the amino acid changes in HA that were linked to a switch of receptor-binding preference from nonhuman to pandemic, as well as pandemic to seasonal. Our data provide insight into why seasonal viruses are associated with reduced alveolar infection and damage and suggest new considerations for designing anti-HA vaccines and drugs. The results provide a better understanding of viral tropism and pathogenesis in humans that will be important for prediction and surveillance of zoonotic, pandemic, and epidemic influenza outbreaks. DATABASE: The novel hemagglutinin nucleotide sequences reported here were deposited in GISAID under the accession numbers of EPI685738 for A/Yamaguchi/20/2006(H1N1) and EPI685740 for A/Kitakyushu/10/2006(H1N1).

Preparation of a Molecular Library of Branched ß-Glucan Oligosaccharides Derived from Laminarin.

To study the structure of ß-glucans, we developed a separation method and molecular library of ß-glucan oligosaccharides. The oligosaccharides were prepared by partial acid hydrolysis from laminarin, which is a ß-glucan of Laminaria digitata. They were labeled with the 2-aminopyridine fluorophore and separated to homogeneity by size-fractionation and reversed phase high-performance liquid chromatography (HPLC). Branching structures of all isomeric oligosaccharides from trimers to pentamers were determined, and a two-dimensional (2D)-HPLC map of the ß-glucan oligosaccharides was made based on the data. Next, structural analysis of the longer ß-glucan oligosaccharide was performed using the 2D-HPLC map. A branched decamer oligosaccharide was isolated from the ß-glucan and cleaved to smaller oligosaccharides by partial acid hydrolysis. The structure of the longer oligosaccharide was successfully elucidated from the fragment structures determined by the 2D-HPLC map. The molecular library and the 2D-HPLC map described in this study will be useful for the structural analysis of ß-glucans.

Highly efficient transglycosylation of sialo-complex-type oligosaccharide using Coprinopsis cinerea endoglycosidase and sugar oxazoline.

OBJECTIVES: To establish an efficient method of chemoenzymatic modification for making N-linked oligosaccharide chains of glycoproteins structurally homogeneous, which crucially affects their bioactivities. RESULTS: Deglycosylated-RNase B (GlcNAc-RNase B; acceptor), sialylglyco (SG)-oxazoline (donor) and an N180H mutant of Coprinopsis cinerea endo-ß-N-acetylglucosaminidase (Endo-CCN180H) were employed. pH 7.5 was ideal for both SG-oxazoline's stability and Endo-CC's transglycosylation reaction. The most efficient reaction conditions for producing glycosylated-RNase B, virtually modified completely with sialo-biantennary-type complex oligosaccharide, were: 80 µg GlcNAc-RNase B, 200 µg SG-oxazoline and 3 µg Endo-CCN180H in 20 µl 20 mM Tris/HCl pH 7.5 at 30 °C for 30-60 min. CONCLUSIONS: This transglycosylation method using SG-oxazoline and Endo-CCN180H is beneficial for producing pharmaceutical glycoproteins modified with homogenous biantennary-complex-type oligosaccharides.

Trisaccharide containing α2,3-linked sialic acid is a receptor for mumps virus.

Mumps virus (MuV) remains an important pathogen worldwide, causing epidemic parotitis, orchitis, meningitis, and encephalitis. Here we show that MuV preferentially uses a trisaccharide containing α2,3-linked sialic acid in unbranched sugar chains as a receptor. Crystal structures of the MuV attachment protein hemagglutinin-neuraminidase (MuV-HN) alone and in complex with the α2,3-sialylated trisaccharide revealed that in addition to the interaction between the MuV-HN active site residues and sialic acid, other residues, including an aromatic residue, stabilize the third sugar of the trisaccharide. The importance of the aromatic residue and the third sugar in the MuV-HN-receptor interaction was confirmed by computational energy calculations, isothermal titration calorimetry studies, and glycan-binding assays. Furthermore, MuV-HN was found to bind more efficiently to unbranched α2,3-sialylated sugar chains compared with branched ones. Importantly, the strategically located aromatic residue is conserved among the HN proteins of sialic acid-using paramyxoviruses, and alanine substitution compromised their ability to support cell-cell fusion. These results suggest that not only the terminal sialic acid but also the adjacent sugar moiety contribute to receptor function for mumps and these paramyxoviruses. The distribution of structurally different sialylated glycans in tissues and organs may explain in part MuV's distinct tropism to glandular tissues and the central nervous system. In the crystal structure, the epitopes for neutralizing antibodies are located around the α-helices of MuV-HN that are not well conserved in amino acid sequences among different genotypes of MuV. This may explain the fact that MuV reinfection sometimes occurs.

A rationally engineered yeast pyruvyltransferase Pvg1p introduces sialylation-like properties in neo-human-type complex oligosaccharide.

Pyruvylation onto the terminus of oligosaccharide, widely seen from prokaryote to eukaryote, confers negative charges on the cell surface and seems to be functionally similar to sialylation, which is found at the end of human-type complex oligosaccharide. However, detailed molecular mechanisms underlying pyruvylation have not been clarified well. Here, we first determined the crystal structure of fission yeast pyruvyltransferase Pvg1p at a resolution of 2.46 Å. Subsequently, by combining molecular modeling with mutational analysis of active site residues, we obtained a Pvg1p mutant (Pvg1p(H168C)) that efficiently transferred pyruvyl moiety onto a human-type complex glycopeptide. The resultant pyruvylated human-type complex glycopeptide recognized similar lectins on lectin arrays as the α2,6-sialyl glycopeptides. This newly-generated pyruvylation of human-type complex oligosaccharides would provide a novel method for glyco-bioengineering.

Occurrence of free sialyl oligosaccharides related to N-glycans (sialyl free N-glycans) in animal sera.

Free oligosaccharides that are structurally related to N-glycans [free N-glycans (FNGs)] are widely distributed in the cytosol of animal cells. The diverse molecular mechanisms responsible for the formation of these FNGs have been well clarified. In this study we demonstrate the wide occurrence of sialylated FNGs in sera of various animals. The features of these extracellular FNGs are quite distinct from the cytosolic FNGs, as they are Gn2-type glycans, bearing an N,N'-diacetylchitobiose unit at their reducing termini, while the cytosolic FNGs are predominantly Gn1-type, with a single GlcNAc at their reducing termini. The major structures observed varied from species to species, and the structures of the FNGs appear to be correlated with the major sialyl N-glycans on serum glycoproteins, suggesting that the serum FNGs are produced by hepatocytes. Interestingly, glycan-profiles of the FNGs indicated that they are altered in a developmental stage-dependent manner. Sialyl FNGs in the sera may not only be of biological relevance, in that they might reflect the functionality of the liver, but also can be attractive sources for obtaining uniform sialyl FNGs in the chemoenzymatic synthesis of glycoproteins.

Cooperative Interactions of Oligosaccharide and Peptide Moieties of a Glycopeptide Derived from IgE with Galectin-9.

We previously showed that galectin-9 suppresses degranulation of mast cells through protein-glycan interaction with IgE. To elucidate the mechanism of the interaction in detail, we focused on identification and structural analysis of IgE glycans responsible for the galectin-9-induced suppression using mouse monoclonal IgE (TIB-141). TIB-141 in combination with the antigen induced degranulation of RBL-2H3 cells, which was almost completely inhibited by human and mouse galectin-9. Sequential digestion of TIB-141 with lysyl endopeptidase and trypsin resulted in the identification of a glycopeptide (H-Lys13-Try3; 48 amino acid residues) with a single N-linked oligosaccharide near the N terminus capable of neutralizing the effect of galectin-9 and another glycopeptide with two N-linked oligosaccharides (H-Lys13-Try1; 16 amino acid residues) having lower activity. Enzymatic elimination of the oligosaccharide chain from H-Lys13-Try3 and H-Lys13-Try1 completely abolished the activity. Removal of the C-terminal 38 amino acid residues of H-Lys13-Try3 with glutamyl endopeptidase, however, also resulted in loss of the activity. We determined the structures of N-linked oligosaccharides of H-Lys13-Try1. The galectin-9-binding fraction of pyridylaminated oligosaccharides contained asialo- and monosialylated bi/tri-antennary complex type oligosaccharides with a core fucose residue. The structures of the oligosaccharides were consistent with the sugar-binding specificity of galectin-9, whereas the nonbinding fraction contained monosialylated and disialylated biantennary complex type oligosaccharides with a core fucose residue. Although the oligosaccharides linked to H-Lys13-Try3 could not be fully characterized, these results indicate the possibility that cooperative binding of oligosaccharide and neighboring polypeptide structures of TIB-141 to galectin-9 affects the overall affinity and specificity of the IgE-lectin interaction.

Preparation of Glycan Arrays Using Pyridylaminated Glycans.

We describe the method to prepare neoglycoproteins from the conjugation of bovine serum albumin and pyridylaminated glycans. Large quantities of glycans (>1 mg) can be pyridylaminated and then converted to their 1-amino-1-deoxy derivatives by reaction with hydrogen followed by hydrazine. These pyridylaminated glycans can then be conjugated to bovine serum albumin via esterification with N-( m-maleimidobenzoyloxy)succinimide to form a neoglycoprotein, e.g., glycosylated bovine serum albumin. As a demonstration, we prepared High-mannose bovine serum albumin, which was immobilized on an activated glass slide. Then, we showed that the neoglycoprotein bind to Cy3-labeled Lens culinaris agglutinin, a mannose-specific plant lectin, as detected using an evanescent-field-activated fluorescence scanner system.

Transglycosylation Activity of Glycosynthase Mutants of Endo-ß-N-Acetylglucosaminidase from Coprinopsis cinerea.

Endo-ß-N-acetylglucosaminidase (ENGase), which catalyzes hydrolysis of N-linked oligosaccharides, is a useful tool for analyzing oligosaccharide contents of glycoproteins. However, there are only a few known ENGases that can catalyze the hydrolysis of human complex type oligosaccharides, and although commercially available, they are expensive. Here, we report the cloning of two ENGase encoding cDNAs from the basidiomycete fungus Coprinopsis cinerea, Endo-CC1 and Endo-CC2. We successfully expressed recombinant His6-tagged Endo-CC1 and Endo-CC2 in Escherichia coli and purified them for enzymatic characterization. Both Endo-CC1 and Endo-CC2 showed hydrolytic activity on high-mannose and complex type oligosaccharides. Since Endo-CC1 could be prepared more easily than Endo-CC2 from E. coli cultures, we examined the enzymatic properties of Endo-CC1 in detail. Our results showed that Endo-CC1 acted on both N-linked high-mannose type and sialobiantennary type complex oligosaccharides of glycoproteins RNase B and human transferrin, respectively, but not on the sialotriantennary type complex oligosaccharide of glycoprotein fetuin. Examination of the transglycosylation activity of Endo-CC1 revealed that the wild-type Endo-CC1 could not transfer the sialobiantennary type complex oligosaccharide onto the deglycosylated RNase B. To obtain an Endo-CC1 mutant with desired transglycosylation activity, we performed mutation analysis of the active site residue Asn 180 (N180), known to be important for catalysis, by individually replacing it with the remaining 19 amino acid residues. Transglycosylation analyses of these mutants led us to identify one mutant, namely Endo-CC1N180H, which exhibited the desired transglycosylation activity. Taken together, we suggest that Endo-CC1 would potentially be a valuable tool for analyzing oligosaccharides on glycoproteins, as large quantities of it could be made available more easily and less expensively than the currently used enzyme, Endo-M.